The present invention relates to genetically modified yeasts for producing glycoproteins having optimized and homogeneous glycan structures. These yeasts comprise inactivation of the Och1 gene, integration by homologous recombination into an auxotrophy marker of an expression cassette comprising a first promoter, and an open reading phase comprising the sequence coding for an α-1-2 mannosidase I, and integration of a cassette comprising a second promoter different from said first promoter and the sequence coding for an exogenous glycoprotein. These yeasts allow production of EPO with optimized and homogeneous 98% glycosylation.
The production of glycoproteins or glycopeptides having glycans of the complex type, i.e. structures identical with oligosaccharides added during post-translational modifications in humans, has been a sought goal for quite a few years in the pharmaceutical industry. Indeed, many studies have shown the importance of oligosaccharides for optimizing the activity of therapeutic glycoproteins or further for improving their half-life time once they are administered. For example, human erythropoietin (HuEPO) is a glycoprotein of 166 amino acids containing three N-glycosylation sites in positions Asn-24, Asn-38 and Asn-83 and an O-glycosylation site of the mucin type in position Ser-126. EPO is a particularly relevant model for studying N-glycosylation because of its glycosylated structures representing 40% of its molecular weight. The EPO molecule considered as natural is the urinary form (uHuEPO) (Takeuchi et al., 1988, Tusda 1988, Rahbeck-nielsen 1997). Recombinant EPO (rHuEPO) is presently produced in CHO cells (Sasaki H et al., Takeuchi et al., 1988) or in BHK cells (Nimtz et al., 1993). The rHuEPOs expressed in cell lines have N-glycan structures different from the structures found in uHuEPO. These differences may have repercussion in vitro (Higuchi et al., 1992; Takeuchi et al., 1990) but seem to be more sensitive in vivo by a drastic loss of activity for the deglycosylated forms and by an increase of activity correlated with the number of sialic acids present on the structure (Higuchi M et al., 1992).
In order to produce glycoproteins having optimum N- or O-glycosylation, many technical solutions have been proposed. Mention may be made of in vitro modifications of glycan structures by adding sugars such as galactose, glucose, fucose or even sialic acid by means of different glycosyl transferases or by suppressing certain sugars such as mannose with mannosidases. This technique is described in WO 03/031464 (Neose). It is however possible to wonder how such a technique may be applied on a large scale since this involves many steps for sequential modification of several given oligosaccharides present on a same glycoprotein. In each step, strict control of the reaction should be carried out and production of homogeneous glycanic structures should be ensured. Now, in the case when many oligosaccharides have to be modified on a glycoprotein, a sequential reaction may result in undesirable and heterogeneous modifications. This technique is therefore not compatible with the preparation of biodrugs. Further, the use of purified enzymes for production on an industrial scale does not seem to represent a viable economical alternative.
The same applies with chemical coupling techniques, such as those described in documents WO 2006/106348 and WO 2005/000862. These chemical coupling techniques involve tedious reactions, protection/deprotection steps, multiple checks. In the case when many oligosaccharides have to be modified on a given glycoprotein, a sequential reaction may also result in undesirable and heterogeneous modifications. Other technologies using mammal cell lines such as YB2/0 described in WO 01/77181 (LFB) or further CHO lines genetically modified in WO 03/055993 (Kyowa) have demonstrated that slight fucosylation of the Fc region of the antibodies improves ADCC activity by a factor 100. However, these technologies specifically relate to the production of antibodies.
Finally, production of glycoproteins in yeasts or filamentous fungi has been proposed by transformation of these micro-organisms with plasmids allowing expression of mannosidases and of different glycosyl transferases. This approach was described in WO 02/00879 (Glycofi). However, to this day, it has not been demonstrated that these micro-organisms are stable over time in a high capacity fermenter for producing clinical batches. Also, it has not been shown that this transformation enables production of glycoproteins with the desired and homogeneous glycans.
With the purpose of producing rHuEPO having N-glycan structures with which optimum activity may be obtained in vivo, we expressed an rHuEPO in genetically modified S. cerevisiae and S. pombe yeasts. These yeasts showed strong expression of rHuEPO having homogeneous and well-characterized N-glycosylation units. In a second phase, we started with genetically modified yeasts and we incorporated other modifications in order to produce more complex N-glycan units, depending on their sialylation levels. The yeast system is known for its capacity of rapidly producing a large amount of proteins but the modified yeasts described hereafter are also capable of N-glycosylation of the produced proteins in a “humanized” and homogeneous way. Further, these yeasts are found to be stable under conditions of production on a large scale. Finally, in the case of mutations leading to genotype reversion, these yeasts are constructed so as to allow them to be restored identically, which is required within the scope of producing clinical batches.
Thus, this is the first example which illustrates targeted integration methods in particular loci which have been used for the whole of the invention, methods allowing control of interrupted and selected genome regions to within one nucleotide, and therefore allowing restoration of the interruption in the case of spontaneous genome reversion. This method should be opposed to the one described in WO 02/00879, consisting of transforming a yeast strain with a bank of sequences and subsequently selecting the best clone without any genomic characterization. Indeed, in WO 02/00879 the integrations are random and the clones are exclusively selected on the basis of the profile of N-glycan structures of the produced proteins, which involves, in the case of mutations, reversion or any other genetic modification, pure and simple loss of the clone of interest. The advantage of the technology according to the invention is to provide increased safety to the user, by providing him/her with a guarantee of controlling, tracking genetic modifications, and especially the possibility of reconstructing a clone which will strictly have the same capacities.
Further, for the first time we provide a “Glycan-on-Demand” technology from the Amélie strain as described hereafter. Under these conditions, the homogeneity of the structures is more important than in the CHO systems which glycosylate like mammals. Indeed, the obtained results (see EPO spectrum), report a glycan structure of the Man5GlcNAc2 type representing about 98% of the N-glycans present on the protein. The system is therefore designed so as to force glycosylation in order to obtain a desired unit in very large proportion. The Amélie strain is the clone used as a basis for elaborating any other strain intended to produce humanized, hybrid or complex glycans, which one wishes to obtain. The advantage of this strain is to form a starting point, which was demonstrated as being a stable and homogeneous system producing 98% of Man5GlcNAc2 glycoproteins, which may be reworked for additional modifications such as the introduction of a GlcNAc transferase, of a fucosyl transferase, of a galactosyl and/or sialyl transferase, on demand, rapidly, according to the desired final structures.